EP3546596A1 - Trousse de détection de dysfonctionnements géniques - Google Patents

Trousse de détection de dysfonctionnements géniques Download PDF

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EP3546596A1
EP3546596A1 EP19151537.8A EP19151537A EP3546596A1 EP 3546596 A1 EP3546596 A1 EP 3546596A1 EP 19151537 A EP19151537 A EP 19151537A EP 3546596 A1 EP3546596 A1 EP 3546596A1
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region
gene
target gene
tmprss2
target
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EP3546596B1 (fr
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Heather R. Sanders
Maher Albitar
Aurelia Meloni-Ehrig
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Quest Diagnostics Investments LLC
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Quest Diagnostics Investments LLC
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • G—PHYSICS
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    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present technology relates generally to detection of gene dysregulations such as those arising from gene fusions and chromosomal abnormalities, which may be associated with various diseases.
  • the present technology relates to the detection of gene dysregulations using multiplex quantitative RT-PCR.
  • Variations in chromosome structure involve changes in parts of chromosomes rather than changes in the number of chromosomes or sets of chromosomes in the genome.
  • mutations There are four common types of mutations: deletions and duplications (both of which involve a change in the amount of DNA on a chromosome), inversions (which involve a change in the arrangement of a chromosomal segment), and translocations (which involve a change in the location of a chromosomal segment). All four classes of chromosomal structure mutations are initiated by one or more breaks in the chromosome. If a break occurs within a gene, then a gene mutation has been produced, the consequence of which depends on the function of the gene and the time of its expression. Wherever the break occurs, the breakage process leaves broken ends, which may adhere to other broken chromosome ends or the normal ends of other chromosomes.
  • the present disclosure provides a method for detecting the presence or absence of a dysregulation in a target gene in a sample.
  • the method may include: (a) measuring the amount of transcription of a 5' region of the target gene and a 3' region of the target gene in the test sample; and (b) comparing the relative expression of the 5' region to the 3' region of the target gene in the test sample to the relative expression of the 5' region to the 3' region of the target gene in a reference sample.
  • the method may also provide that a difference in the relative expression in the test sample compared to the reference sample is indicative of the presence of a gene dysregulation.
  • the relative amount of transcript can be determined using real-time PCR and comparing the threshold cycle, or Ct, value, for each amplicon. The Ct value can be normalized to a reference sample.
  • the combination of the inventive methods with comparative hybridization will be able to detect both balanced and unbalanced rearrangements and provide a more accurate diagnosis than if the comparative hybridization technique was used alone.
  • the comparative hybridization technique may be used as a confirmatory assay.
  • target gene dysregulations may arise from gene fusions and chromosomal abnormalities including, for example, translocations, deletions, inversions, and insertions.
  • Any cancer or other disorder associated with a gene dysregulation may be diagnosed using any of the foregoing methods.
  • Disorders suitable for diagnosis include, for example, pediatric soft tissue sarcomas that have indeterminate histologies.
  • the biological sample is contacted with the one or more 5' target primer pairs and the one or more 3' target primer in a multiplex amplification reaction.
  • the detecting is accomplished using a labeled oligonucleotide probe complementary to each amplification product.
  • each oligonucleotide probe may include a different detectable label, such as a donor fluorophore and quencher moiety.
  • at least one of the primers for the 5' region and/or at least one of the primers for the 3' region is detectably labeled, preferably with different detectable labels.
  • the amplifying is performed using quantitative RT-PCR, e.g. , real-time RT-PCR.
  • the chromosomal abnormality is selected from the group consisting of: a translocation, a deletion, an inversion, and an insertion.
  • the biological sample is a sample from a subject to be tested for a chromosomal abnormality.
  • the methods further include amplifying a region of an endogenous control gene transcript present in the biological sample with a primer pair complementary to the endogenous control gene and detecting the amplification of the region of the endogenous control gene.
  • the amount of amplified target gene transcripts i.e., the 5' region and the 3' region
  • Suitable endogenous control genes include, for example, ABL.
  • the method further includes: (a) measuring the amount of transcription of a 5' region of a second target gene and a 3' region of the second target gene in the test sample; and (b) comparing the relative expression of the 5' region to the 3' region of the second target gene in the test sample to the relative expression of the 5' region to the 3' region of the second target gene in a reference sample.
  • the method may also provide that a difference in the relative expression of both the target gene and the second target gene in the test sample compared to the reference sample is indicative of the presence of a target gene:second target gene translocation.
  • Exemplary target gene:and second target gene translocations include TMPRSS2:ERG, TMPRSS2:ETV1, and EML4:ALK.
  • the disclosure provides a kit for detecting a genetic abnormality in a sample.
  • the kit may include: (a) at least one oligonucleotide for determining the level of expression of at least one sequence from the 5' region of a target gene; and (b) at least one oligonucleotide for determining the level of expression of at least one sequence from the 3' region of the target gene.
  • the target gene is TMPRSS2 or ALK.
  • the kits further include one or more reagents for performing real-time RT-PCR.
  • Chromosomal abnormalities include, for example, translocations, deletions and insertions.
  • the term "detecting” refers to observing a signal from a detectable label to indicate the presence of a target nucleic acid in the sample.
  • the term detecting does not require the method to provide 100% sensitivity and/or 100% specificity.
  • "sensitivity” is the probability that a test is positive, given that the subject has a target nucleic acid sequence
  • specificity is the probability that a test is negative, given that the subject does not have the target nucleic acid sequence.
  • a sensitivity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred.
  • nucleic acids of the present invention include, for example, inosine and 7-deazaguanine.
  • Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.
  • Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
  • Complementarity may be "partial” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete,” “total,” or “full” complementarity between the nucleic acids.
  • detectable label refers to a molecule or a compound or a group of molecules or a group of compounds associated with a probe and is used to identify the probe hybridized to a genomic nucleic acid or reference nucleic acid. In some cases, the detectable label may be detected directly. In other cases, the detectable label may be a part of a binding pair, which can then be subsequently detected. Signals from the detectable label may be detected by various means and will depend on the nature of the detectable label.
  • means to detect detectable label include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means.
  • a "fragment" in the context of a gene fragment or a chromosome fragment refers to a sequence of nucleotide residues which are at least about 10 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 250 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 10,000 nucleotides, at least about 20,000 nucleotides, at least about 50,000 nucleotides, at least about 100,000 nucleotides, at least about 500,000 nucleotides, at least about 1,000,000 nucleotides or more.
  • genetic abnormality refers to a deviation of the nucleic acid sequence from a wild-type or normal genetic sequence.
  • a genetic abnormality may reflect a difference between the full genetic complement of an organism, or any portion thereof, as compared to a normal full genetic complement of all chromosomes in that organism.
  • a genetic abnormality may include a change in chromosomes or a portion thereof (e.g ., deletions, duplications, amplifications); or a change in chromosomal structure ( e.g ., translocations, point mutations).
  • Genetic abnormality may be hereditary, i.e. , passed from generation to generation or non-hereditary. Genetic abnormalities may be present in some cells of an organism or in all cells of that organism.
  • endogenous control gene refers to genes that are generally always expressed and thought to be involved in routine cellular metabolism. Endogenous control genes are well known and include such genes as ABL, glyceraldehyde-3-phosphate dehydrogenase (G3PDH or GAPDH), albumin, actins, tubulins, cyclophilin, hypoxanthine phosphoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs. Detection of endogenous control genes in a diagnostic assay may serve as a positive control for the assay.
  • G3PDH or GAPDH glyceraldehyde-3-phosphate dehydrogenase
  • HRPT hypoxanthine phosphoribosyltransferase
  • isolated refers to molecules, such as nucleic acid, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • An isolated molecule is therefore a substantially purified molecule.
  • oligonucleotide refers to a short polymer composed of deoxyribonucleotides, ribonucleotides or any combination thereof. Oligonucleotides are generally between about 10, 11, 12, 13, 14, 15, 20, 25, or 30 to about 150 nucleotides (nt) in length, more preferably about 10, 11, 12, 13, 14, 15, 20, 25, or 30 to about 70 nt, and most preferably between about 18 to about 26 nt in length.
  • a "forward primer” is a primer that is complementary to the anti-sense strand of dsDNA.
  • a “reverse primer” is complementary to the sense-strand of dsDNA.
  • a "5' target primer pair” is at least one forward primer and at least one reverse primer that amplifies the 5' region of a target nucleotide sequence.
  • a "3' target primer pair” is at least one forward primer and at least one reverse primer that amplifies the 3' region of a target nucleotide sequence.
  • oligonucleotide e.g ., a probe or a primer
  • hybridization or “hybridizing” refers to the process by which an oligonucleotide single strand anneals with a complementary strand through base pairing under defined hybridization conditions. It is a specific, i.e. , non-random, interaction between two complementary polynucleotides.
  • Hybridization and the strength of hybridization is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the T m of the formed hybrid.
  • an oligonucleotide is "specific" for a nucleic acid if the oligonucleotide has at least 50% sequence identity with a portion of the nucleic acid when the oligonucleotide and the nucleic acid are aligned.
  • An oligonucleotide that is specific for a nucleic acid is one that, under the appropriate hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and more preferably at least 98% sequence identity.
  • Sequence identity can be determined using a commercially available computer program with a default setting that employs algorithms well known in the art (e.g. , BLAST).
  • sequences that have "high sequence identity” have identical nucleotides at least at about 50% of aligned nucleotide positions, preferably at least at about 60% of aligned nucleotide positions, and more preferably at least at about 75% of aligned nucleotide positions.
  • target nucleic acid refers to nucleic acid sequence which is intended to be identified.
  • Target nucleic acids may include 5' or 3' regions of a target gene or any other sequence of interest.
  • Target nucleic acids may represent alternative sequences or alleles of a particular gene.
  • Target nucleic acids can be double stranded or single stranded, or partially double stranded, or partially single stranded or a hairpin molecule.
  • Target nucleic acids can be about 1-5 bases, about 10 bases, about 20 bases, about 50 bases, about 100 bases, about 500 bases, about 1,000 bases, about 2,000 bases, 2,500 bases, about 3,000 bases, about 3,000 bases, about 4,000 bases, about 5,000 bases, about 7,500 bases, about 10,000 bases, about 20,000 bases, about 30,000 bases, about 40,000 bases, about 50,000 bases, about 75,000 bases, about 100,000 bases, about 1,000,000 bases or more.
  • RNA when referring to a target nucleic acid, refers to any nucleic acid that is representative of the genomic nucleic acid of a cell including, for example, RNA in any form (e.g., mRNA, pre-mRNA, and snRNA) and synthetic representations of such as cDNA.
  • test sample refers to a sample, which contains nucleic acid or is suspected of containing nucleic acid.
  • the nucleic acids in the test sample are for use in accordance with the methods disclosed herein.
  • a test sample is a biological sample.
  • the term "subject” refers to a mammal, such as a human, but can also be another animal such as a domestic animal (e.g ., a dog, cat, or the like), a farm animal (e.g. , a cow, a sheep, a pig, a horse, or the like) or a laboratory animal (e.g. , a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like).
  • the term "patient” refers to a "subject” who is, or is suspected to be, afflicted with disease related to a chromosomal abnormality.
  • test samples disclosed herein are represented by, but not limited in any way to, e.g. , blood (or a fraction of blood such as plasma, serum, or particular cell fractions), lymph, mucus, tears, saliva, cystic fluid, urine, semen, stool, CSF, ascites fluid, whole blood, and biopsy samples of body tissue, fine needle aspirate (FNA), bronchalveolar lavage (BAL).
  • FNA fine needle aspirate
  • BAL bronchalveolar lavage
  • This disclosure is also drawn, inter alia , to methods of diagnosing or monitoring cancer.
  • the cancer can be lung or prostate cancer bone and soft tissue sarcomas, various leukemias and lymphomas.
  • the technology generally provides for the detection, measuring, and comparison of gene expression of different regions of a target gene within a test sample. Accordingly, the various aspects relate to the collection, preparation, separation, identification, characterization, and comparison of the abundance of messenger RNA in a test sample.
  • the technology further relates to detecting and/or monitoring a sample containing a messenger RNA for a 5' region of a target gene and a 3' region of a target gene.
  • the phrases "detecting the amount” or “detecting the level” refer to the quantity of transcript from any gene or part of a gene, such as the 5' region of a gene, a 3' region of the target gene, a reference gene.
  • the amount can be expressed as a concentration, as a number of copies, or as a Ct value, for example.
  • the threshold cycle, or Ct, value is the cycle at which signal intersects a threshold value when performing real-time nucleic acid amplification.
  • Specimens that do not contain a chromosomal abnormality within a target gene will demonstrate the same expression pattern, between the 5' region and the 3' region because they are linked in a unimolecular fashion.
  • the 5' and 3' regions may show independent expression patterns for the 5' and 3' regions.
  • the 5' and 3' regions will show different expression patterns because these two regions are now unlinked on the chromosome.
  • a gene that undergoes certain rearrangements will exhibit differential expression of the 5' region relative to the 3' region. This occurs in situations where the 5' region of a gene remains under the control of the gene's regulatory elements, e.g. , those elements contained in the 5' untranslated region (UTR).
  • the 3' region of the gene is juxtaposed so as to be under the control of different regulatory elements or none at all.
  • the 5' region of the gene e.g. , at least one sequence that is specific to the 5' region such that it occurs upstream of the mutation break point or deletion site
  • the 3' region e.g.
  • At least one sequence that is specific to the 3' region of the gene that occurs downstream of the mutation break point or deletion site) will not be expressed, in the case where the 3' region is deleted, or translocated to a position that is not actively expressed, or expressed at a level consistent with the regulatory elements of a different gene.
  • the methods provide for detecting these mutations that result in the differential expression of the 5' region of a gene relative to the 3' region of the gene.
  • This situation occurs in many prostate cancer patients, who have a translocation of the TMPRSS2 gene such that the 5' region of the TMPRSS2 gene remains under the control of the robust TMPRSS2 promoter, and the 3' region of the TMPRSS2 gene is translocated such that it is expressed by the less robust ERG or ETV promoter.
  • equal quantity it is meant that the measured amounts of transcript or detectable signal (which correlates to the amount of transcript) for the 5' region and the 3' region do not exhibit a statistically significant difference from the same comparison in control samples.
  • Methods for comparing these values are known to those of skill in the art and include, but are not limited to, a Student's t -test and ANOVA analysis. The artisan recognizes that, because of technical differences inherent in the detection methodologies used herein, the amount of detectable signal from the 5'-region may not necessarily be equal to the amount of detectable signal from the 3'-region even though no chromosomal abnormality is present (i.e., both regions remain linked in a unimolecular manner and under the control of the same regulatory elements).
  • IDE Score 2 ⁇ Ct 3 ⁇ ⁇ target gene ⁇ Ct 5 ⁇ ⁇ target gene wherein the Ct (threshold cycle) values can be obtained by RT-PCR.
  • the 3'- and 5'-target gene measurements may be normalized to an endogenous control gene when calculating an IDE score.
  • Some useful formulae include, for example:
  • the measured amount of the 3'- and 5'-transcripts in the test sample may be normalized to the level of the same transcripts from a control sample, rather than an endogenous gene.
  • the mean amount of transcript or detectable signal for the 5' region and the 3' region are within about 1 standard deviation, within about 0.5 standard deviations, within about 0.2 standard deviations, within about 0.1 standard deviations, or within about 0.01 standard deviations, then there may be no significant difference between the two amounts. In this example, one could conclude that the 5' and 3' regions are expressed in a unimolecular fashion and there is no chromosomal abnormality in the target gene.
  • the mean amount of transcript or detectable signal for the 5' region and the 3' region exceed about 1 standard deviation, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations, then there may be a significant difference between the two amounts.
  • the measured amounts of transcript or detectable signal may be expressed as a "relative amount” or "ratio" of the expression of the 5' region of the target gene relative to the 3' region of the target gene.
  • Relative amounts may be a single value or a range of values. For example, a range of values may be used to generate a standard curve relationship between the relative amount of detectable signal formed versus some other quantity (e.g ., number of mRNA molecules). If the ratio of the expression of the 5' region of the target gene relative to the expression of the 3' region of the target gene is statistically less than or greater than 1, then a chromosomal abnormality is detected.
  • a sample obtained from a subject is assayed to determine the relative expression levels of the 5' and 3' regions of a particular gene or nucleic acid sequence of interest.
  • Real-time RT-PCR real-time reverse transcription-polymerase chain reaction
  • RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell.
  • oligonucleotide primers and probes that are used to detect differential 5' and 3' expression from any gene of interest, provided the sequence of the gene of interest is known.
  • the size of the primer will depend on many factors, including the ultimate function or use of the oligonucleotide.
  • An oligonucleotide that functions as an extension primer or probe, for example, will be sufficiently long to prime the synthesis of extension products in the presence of a catalyst, e.g. , DNA polymerase, and deoxynucleotide triphosphates.
  • an insertion or transposition event can lead to the differential expression of the 5' region and the 3' region of a target gene.
  • the insertion of, for example, a promoter or other regulatory element, or the transposition of a transposable element into the middle of the coding sequence of a gene of interest can create a situation where the 5' region of the target gene is expressed at a different level than the 3' region of the target gene.
  • any such mutation that results in the differential expression of a 5' region of a target gene and the 3' region of the target gene is detectable according to the methods, compositions and kits described herein.
  • One of skill in the art would know how to directed, for example, RT-PCR primers to a 5' region of a gene of interest that occurs at or near the start of transcription, thereby ensuring a product corresponding to a 5' region that occurs downstream of a potential chromosomal abnormality.
  • One of skill in the art need only refer to the known sequence of the target gene and known base-pairing rules to determine an effective RT-PCR primer or primer pair.
  • one of skill in the art could design a primer or primer pair directed to a 3' region of the gene of interest.
  • Genetic duplication is any duplication of a region of the genomic sequence. It may occur as an error in homologous recombination, a retrotransposition event, or duplication of an entire chromosome. Duplication of a gene has been associated with several diseases such as some cases of pagetic osteosarcoma is associated with duplication of MYC gene ( Sarcoma, 1(3-4):131-134, 1997 ), some cases of breast cancer are associated with duplication of HER-2/neu gene ( Ann Oncol., 12(suppl 1):S3-S8, 2001 ), some cases of bladder tumor are associated with duplication of c-erb-2 gene ( Cancer Res., 55:2422-2430, 1995 ).
  • This translocation fuses the BCR gene locus of chromosome 22 and the proto-oncogene ABL locus of chromosome 9 to form a bcr/abl oncogenic protein ( Tefferi et al. Mayo Clin Proc, 80(3):390-402, 2005 ).
  • Philadelphia chromosome was first associated with CML, it is now known to be an indicator of prognosis in other blood disorders such as acute lymphoblastic leukemia (ALL).
  • ALL acute lymphoblastic leukemia
  • Translocations have been linked with other diseases.
  • the fusion of the CBP gene of chromosome 16 to the MLL gene of chromosome 11 through a translocation between chromosomes 11 and 16 has been associated with leukemia ( Zhang et al., Genes Chromosomes Cancer, 41(3):257-65, 2004 ).
  • a translocation between chromosomes 8 and 21, resulting in a fusion of the AML1 and ETO genes is involved in nearly 15 % of acute myeloid leukemia (AML) cases ( Zhang et al., Science, 305:1286-9, 2004 ).
  • AML acute myeloid leukemia
  • TMPRSS2 a fusion of the androgen-regulated gene TMPRSS2 and members of the ETS family of transcription factors (e.g., ERG, ETV1, and ETV4) have been identified in prostate cancers.
  • ERG 21q22.3
  • ETV1 (7p21.2 ETV4 (17q21).
  • Genetic abnormalities may also be point mutations insertions, or deletions.
  • a point mutation, or substitution is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide. Insertion and deletion includes insertions or deletions of a single base pair. Mutations in the gene or chromosome often are associated with diseases such as sickle cell anemia, cystic fibrosis, hemophilia, phenylketonuria, spina bifida, etc.
  • the methods of the present invention relate to the detection of chromosomal abnormalities in a target gene by amplifying a 5' region of the target gene transcript, if present, in a biological sample with one or more 5' target primer pairs which are complementary to the 5' region of the target gene; and amplifying a 3' region of the target gene transcript, if present, in the biological sample with one or more 3' target primer pairs which are complementary to the 3' region of the target gene.
  • Such regions can be amplified and isolated by PCR using oligonucleotide primers designed based on genomic and/or cDNA sequences that encompass the regions. Any target gene that is potentially affected by chromosomal abnormalities could be assayed according to the methods described herein.
  • the term "5' region” refers to the portion of a polynucleotide located towards the 5' end of the polynucleotide relative to the 3' region, and may or may not include the 5' most nucleotide(s) of the same polynucleotide.
  • the 5'-region refers to a region that is in the 5' direction or upstream of a translocation breakpoint.
  • the 5' region may be located near the 5' end of the transcribed portion of the target gene.
  • the 5' region encompasses all or a portion of the 5' untranslated region (UTR) of the target gene.
  • the 5' region is located downstream of the start codon (if the target gene is a protein-coding gene); for example, at least 10, at least 50, at least 100, at least 200, or at least 500 nucleotides downstream of the stop codon.
  • the size of the 5' region to be amplified can vary depending on the detection method chosen.
  • the primers may be selected to amplify at least 10, at least 20, at least 30, at least 50, at least 100, at least 200, or at least 500 nucleotides in the 5' region.
  • 3' region refers to the portion of a polynucleotide located towards the 3' end of the polynucleotide relative to the 5' region, and may or may not include the 3' most nucleotide(s) of the same polynucleotide.
  • the 3'-region refers to a region that is in the 3' direction or downstream of a translocation breakpoint.
  • the 3' region may be located near the 3' end of the transcribed portion of the target gene. In some embodiments, the 3' region encompasses all or a portion of the 3' UTR of the target gene.
  • the 3' region is located upstream of the stop codon (if the target gene is a protein-coding gene); for example, at least 10, at least 50, at least 100, at least 200, or at least 500 nucleotides upstream of the stop codon.
  • the size of the 3' region to be amplified can vary depending on the detection method chosen.
  • the primers may be selected to amplify at least 10, at least 20, at least 30, at least 50, at least 100, at least 200, or at least 500 nucleotides in the 3' region.
  • Specimens from which target nucleic acids can be detected and quantified with the methods of the present invention may be obtained from subjects according to methods known to those of skill in the art. Specimens may be taken from body tissue and fluids such as blood (including whole blood, serum, and plasma), urine, cerebrospinal fluid (CSF), synovial fluid, pleural fluid, pericardial fluid, intraocular fluid, tissue biopsies or endotracheal aspirates, sputum, stool, swabs from, e.g ., skin, inguinal, nasal and/or throat.
  • blood including whole blood, serum, and plasma
  • CSF cerebrospinal fluid
  • synovial fluid pleural fluid
  • pericardial fluid intraocular fluid
  • tissue biopsies or endotracheal aspirates e.g ., skin, inguinal, nasal and/or throat.
  • test samples and reference samples are well known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, drawing of blood or other fluids, surgical or needle biopsies, collection of paraffin embedded tissue, collection of body fluids, collection of stool, and the like.
  • the test sample may be obtained from an individual who is suspected of having a disease or a genetic abnormality.
  • specimens are tissue samples (biopsy samples) from a subject having or suspected of having a disease or a genetic abnormality.
  • the nucleic acid may be isolated from the sample according to any methods well known to those of skill in the art. If necessary, the sample may be collected or concentrated by centrifugation and the like. The cells of the sample may be subjected to lysis, such as by treatments with enzymes, heat surfactants, ultrasonication or combinations thereof. The lysis treatment is performed in order to obtain a sufficient amount of RNA derived from the cells of interest, if present in the sample, to detect using RT-PCR. Nucleic acid need not be extracted, but may be made available by suitable treatment of cells or tissue such as described in US Patent Publication No. 2008/131876 .
  • RNA or cDNA generated from mRNA or total RNA may be used.
  • Various methods of RNA extraction are suitable for isolating the RNA. Suitable methods include phenol and chloroform extraction. See Maniatis et al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, page 16.54 (1989 ).
  • kits for isolating mRNA and synthesizing cDNA are commercially available e.g ., RNeasy Protect Mini kit, RNeasy Protect Cell Mini kit from Qiagen.
  • a dual RNA/DNA isolation method employing a trizol based reagent for initial isolation of RNA and DNA from patient samples.
  • the phenol and high salt reagents in the trizol effectively inactivate any disease agent or secondary disease agent that may be present in the patient sample.
  • a silica based column may be used to further isolate the RNA and DNA.
  • the use of silica based columns allows for wash steps to be performed quickly and efficiently while minimizing the possibility of contamination.
  • the wash steps may be used to remove PCR and RT-PCR inhibitors.
  • the column method for nucleic acid purification is advantageous as it can be used with different types of patient samples and the spin and wash steps effectively remove PCR or RT-PCR inhibitors.
  • Nucleic acid samples or target nucleic acids may be amplified by various methods known to the skilled artisan.
  • PCR is used to amplify nucleic acids of interest. Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleotide triphosphates are added to a reaction mixture along with a DNA polymerase, e.g. , Taq polymerase.
  • a DNA polymerase e.g. , Taq polymerase.
  • the target nucleic acids are amplified in a multiplex amplification reaction.
  • multiplex amplification strategies are known in the art and may be used with the methods of the invention.
  • the multiplex amplification strategy may use PCR, RT-PCR or a combination thereof depending on the type of nucleic acid contained in the disease agent(s). For example, if an RNA genome is present, RT-PCR may be utilized.
  • the PCR enzyme may be an enzyme with both a reverse transcription and polymerase function. Furthermore, the PCR enzyme may be capable of "hot start" reactions as is known in the art.
  • the primers will bind to the sequence and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides.
  • the extended primers will dissociate from the target nucleic acid to form reaction products, excess primers will bind to the target nucleic acid and to the reaction products and the process is repeated, thereby generating amplification products. Cycling parameters can be varied, depending on the length of the amplification products to be extended.
  • An internal positive amplification control (IC) can be included in the sample, utilizing oligonucleotide primers and/or probes.
  • Amplification of nucleic acids can be detected by any of a number of methods well-known in the art such as gel electrophoresis, column chromatography, hybridization with a probe, sequencing, melting curve analysis, or "real-time" detection.
  • the amplicon(s) could be detected by first size-separating the amplicons, then detecting the size-separated amplicons.
  • the separation of amplicons of different sizes can be accomplished by, for example, gel electrophoresis, column chromatography, or capillary electrophoresis. These and other separation methods are well-known in the art.
  • amplicons of about 10 to about 150 base pairs whose sizes differ by 10 or more base pairs can be separated, for example, on a 4% to 5% agarose gel (a 2% to 3% agarose gel for about 150 to about 300 base pair amplicons), or a 6% to 10% polyacrylamide gel.
  • the separated nucleic acids can then be stained with a dye such as ethidium bromide and the size of the resulting stained band or bands can be compared to a standard DNA ladder.
  • two or more fragments of interest are amplified in separate reaction vessels. If the amplification is specific, that is, one primer pair amplifies for one fragment of interest but not the other, detection of amplification is sufficient to distinguish between the two types - size separation would not be required.
  • amplified nucleic acids are detected by hybridization with a specific probe.
  • Probe oligonucleotides, complementary to a portion of the amplified target sequence may be used to detect amplified fragments.
  • Hybridization may be detected in real time or in non-real time.
  • Amplified nucleic acids for each of the target sequences may be detected simultaneously ( i.e. , in the same reaction vessel) or individually ( i.e. , in separate reaction vessels).
  • the amplified DNA is detected simultaneously, using two or more distinguishably-labeled, gene-specific oligonucleotide probes, one which hybridizes to the first target sequence and one which hybridizes to the second target sequence.
  • the probe may be detectably labeled by methods known in the art.
  • Useful labels include, e.g. , fluorescent dyes (e.g. , Cy5®, Cy3®, FITC, rhodamine, lanthamide phosphors, Texas red, FAM, JOE, Cal Fluor Red 610®, Quasar 670®), 32 P, 35 S, 3 H, 14 C, 125 I, 131 I, electron-dense reagents ( e.g. , gold), enzymes, e.g. , as commonly used in an ELISA ( e.g.
  • label include ligands or oligonucleotides capable of forming a complex with the corresponding receptor or oligonucleotide complement, respectively.
  • the label can be directly incorporated into the nucleic acid to be detected, or it can be attached to a probe ( e.g. , an oligonucleotide) that hybridizes or binds to the nucleic acid to be detected.
  • One general method for real time PCR uses fluorescent probes such as the TaqMan® probes, molecular beacons, and ScorpionsTM. Real-time PCR quantitates the initial amount of the template with more specificity, sensitivity and reproducibility, than other forms of quantitative PCR, which detect the amount of final amplified product. Real-time PCR does not detect the size of the amplicon.
  • the probes employed in ScorpionTM and TaqMan® technologies are based on the principle of fluorescence quenching and involve a donor fluorophore and a quenching moiety.
  • quencher moiety means a molecule that, in close proximity to a donor fluorophore, takes up emission energy generated by the donor and either dissipates the energy as heat or emits light of a longer wavelength than the emission wavelength of the donor. In the latter case, the quencher is considered to be an acceptor fluorophore.
  • the quenching moiety can act via proximal ( i.e. , collisional) quenching or by Forster or fluorescence resonance energy transfer (“FRET"). Quenching by FRET is generally used in TaqMan® probes while proximal quenching is used in molecular beacon and ScorpionTM type probes.
  • U.S. Patent Nos. 5,652,099 and 6,268,132 also describe nucleoside analogs for incorporation into nucleic acids, e.g ., DNA and/or RNA, or oligonucleotides, via either enzymatic or chemical synthesis to produce fluorescent oligonucleotides.
  • U.S. Patent No. 5,135,717 describes phthalocyanine and tetrabenztriazaporphyrin reagents for use as fluorescent labels.
  • ScorpionTM probes sequence-specific priming and PCR product detection is achieved using a single molecule.
  • the ScorpionTM probe maintains a stem-loop configuration in the unhybridized state.
  • the fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end.
  • the 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer.
  • the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed.
  • a specific target is amplified by the reverse primer and the primer portion of the ScorpionTM, resulting in an extension product.
  • a fluorescent signal is generated due to the separation of the fluorophore from the quencher resulting from the binding of the probe element of the ScorpionTM to the extension product.
  • TaqMan® probes use the fluorogenic 5' exonuclease activity of Taq polymerase to measure the amount of target sequences in cDNA samples.
  • TaqMan® probes are oligonucleotides that contain a donor fluorophore usually at or near the 5' base, and a quenching moiety typically at or near the 3' base.
  • the quencher moiety may be a dye such as TAMRA or may be a non-fluorescent molecule such as 4-(4 - dimethylaminophenylazo) benzoic acid (DABCYL).
  • melting curve analysis may be used to detect an amplification product.
  • Melting curve analysis involves determining the melting temperature of nucleic acid amplicon by exposing the amplicon to a temperature gradient and observing a detectable signal from a fluorophore. Melting curve analysis is based on the fact that a nucleic acid sequence melts at a characteristic temperature called the melting temperature (T m ), which is defined as the temperature at which half of the DNA duplexes have separated into single strands.
  • T m characteristic temperature
  • the melting temperature of a DNA depends primarily upon its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher T m than those having an abundance of A and T nucleotides.
  • the fluorescent dye may emit a signal that can be distinguished from a signal emitted by any other of the different fluorescent dyes that are used to label the oligonucleotides.
  • the fluorescent dye for determining the melting temperature of a nucleic acid may be excited by different wavelength energy than any other of the different fluorescent dyes that are used to label the oligonucleotides.
  • the second fluorescent dye for determining the melting temperature of the detected nucleic acid is an intercalating agent.
  • Suitable intercalating agents may include, but are not limited to SYBRTM Green 1 dye, SYBRTM dyes, Pico Green, SYTO dyes, SYTOX dyes, ethidium bromide, ethidium homodimer-1, ethidium homodimer-2, ethidium derivatives, acridine, acridine orange, acridine derivatives, ethidium-acridine heterodimer, ethidium monoazide, propidium iodide, cyanine monomers, 7-aminoactinomycin D, YOYO-1, TOTO-1, YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1, JOJO-1, cyanine dimers, YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3, TO-PRO-5, PO-PRO-1, BO-PRO-1, PO-PRO-3, BO-PRO-3,
  • each of the amplified target nucleic acids may have different melting temperatures.
  • each of these amplified target nucleic acids may have a melting temperature that differs by at least about 1°C, more preferably by at least about 2°C, or even more preferably by at least about 4°C from the melting temperature of any of the other amplified target nucleic acids.
  • the methods described herein provide for diagnosing prostate cancer or a susceptibility to cancer in a subject.
  • diagnosis refers to the act or process of identifying or determining a disease or condition in an organism or the cause of a disease or condition by the evaluation of the signs and symptoms of the disease or disorder.
  • a diagnosis of a disease or disorder is based on the evaluation of one or more factors and/or symptoms that are indicative of the disease. That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or condition.
  • Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease, i.e. , there may be differential diagnoses that can be inferred from a diagnostic factor or symptom. Likewise, there may be instances where a factor or symptom that is indicative of a particular disease is present in an individual that does not have the particular disease.
  • the methods include, but are not limited to, prostate and lung cancer and translocations, insertions, inversions and deletions associated with those cancers.
  • the expression level of the 5' region of the TMPRSS2 gene is compared to the expression level of the 3' region of the TMPRSS2 gene in a sample from a subject, wherein a difference in the expression levels of the 5' region of the TMPRSS2 gene and the 3' region of the TMPRSS2 gene is indicative of prostate cancer or a susceptibility to prostate cancer in the subject.
  • the methods described herein provide a prognosis for cancer or in a subject.
  • prognosis refers to a prediction of the probable course and outcome of a clinical condition or disease.
  • a prognosis of a patient is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease.
  • prognosis does not refer to the ability to predict the course or outcome of a condition with 100% accuracy.
  • prognosis refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition.
  • a prognosis may be expressed as the amount of time a patient can be expected to survive.
  • a prognosis may refer to the likelihood that the disease goes into remission or to the amount of time the disease can be expected to remain in remission.
  • Prognosis can be expressed in various ways; for example prognosis can be expressed as a percent chance that a patient will survive after one year, five years, ten years or the like.
  • prognosis may be expressed as the number of years, on average that a patient can expect to survive as a result of a condition or disease.
  • the prognosis of a patient may be considered as an expression of relativism, with many factors affecting the ultimate outcome.
  • prognosis can be appropriately expressed as the likelihood that a condition may be treatable or curable, or the likelihood that a disease will go into remission, whereas for patients with more severe conditions prognosis may be more appropriately expressed as likelihood of survival for a specified period of time.
  • the methods include, but are not limited to, prostate and lung cancer.
  • a prognosis is often determined by examining one or more prognostic factors or indicators. These are markers, such as the presence of a particular chromosomal translocation, the presence or amount of which in a patient (or a sample obtained from the patient) signal a probability that a given course or outcome will occur.
  • markers such as the presence of a particular chromosomal translocation, the presence or amount of which in a patient (or a sample obtained from the patient) signal a probability that a given course or outcome will occur.
  • associating a prognostic indicator with a predisposition to an adverse outcome may involve statistical analysis.
  • the expression level of the 5' region of the TMPRSS2 gene is compared to the expression level of the 3' region of the TMPRSS2 gene in a sample from a subject, wherein a difference in the expression levels of the 5' region of the TMPRSS2 gene and the 3' region of the TMPRSS2 gene is indicative of stage, severity or outcome of prostate cancer in the subject.
  • Nam et al., Br J. Cancer, 97:16390-1695, 2007 examined prostate cancer specimens from 165 patients who underwent surgery for clinically localized prostate cancer between 1998 and 2006.
  • TMPRSS2:ERG gene fusion product They tested for the presence of TMPRSS2:ERG gene fusion product and conducted a survival analysis to determine the prognostic significance of the presence of the TMPRSS2:ERG fusion gene on the risk of prostate cancer recurrence, adjusting for the established prognostic factors.
  • the subgroup of patients with the fusion protein had a significantly higher risk of recurrence (58.4% at 5 years) than did patients who lacked the fusion protein (8.1%, P ⁇ 0.0001).
  • the expression of TMPRSS2:ERG fusion gene is a strong prognostic factor and is independent of grade, stage and PSA level. As such, the present methods are useful in providing a prognosis for recurrence of prostate cancer.
  • the examples below illustrate a standard protocol for performing RT-PCR and analyzing in real time.
  • the TaqMan system of probe labeling is an exemplary method of real time detection of PCR amplicons.
  • the following examples serve to illustrate the present invention and is in no way intended to limit the scope of the invention.
  • ETS E-Twenty Six
  • ETS E-Twenty Six
  • ETV ETS Translocation Variants
  • TMPRSS2:ERG and TMPRSS2:ETV translocations generally result in expression of a fusion transcript containing at minimum the 5' untranslated region of TMPRSS2 fused to coding regions of ERG and ETV.
  • a real-time RT-PCR assay was designed to separately analyze expression levels of the 5' and 3' regions of TMPRSS2 ( FIG. 1 ). Samples that do not contain a translocation involving TMPRSS2 demonstrate the same expression pattern between the 5' and 3' region because they are linked and under the control of the same regulatory elements (e.g., those contained in the 5' untranslated region) ( FIG. 1 , Panel A).
  • TMPRSS2 Distinct 3'-TMPRSS2 expression levels expected to be found in samples containing TMPRSS2 translocations and those without translocations can be established by normalizing the expression levels of 3' TMPRSS2 to 5' TMPRSS2.
  • FIG. 2 shows a dot plot of 25 formalin-fixed and paraffin-embedded (FFPE) tissue samples grouped by TMPRSS2:ERG fusion status.
  • FFPE formalin-fixed and paraffin-embedded
  • RNA from formalin-fixed and paraffin-embedded (FFPE) tissue was extracted by column purification (HighPure miRNA isolation kit, Roche) followed by DNase I digestion (Invitrogen).
  • Plasma RNA was extracted as follows: Study 1: 1 mL plasma from each donor was extracted by NucliSENS® easyMAG® (Biomerieux) followed by DNase I digestion (Invitrogen). Plasma extraction was further optimized in Study 2 as follows: 2 mL plasma from each donor was extracted by NucliSENS® easyMAG® (Biomerieux) followed by DNase I digestion in conjunction with RNA concentration utilizing RNeasy mini kit (Qiagen).
  • Real-time RT-PCR TaqMan primer and probe sets were designed to independently amplify 5' and 3' regions of each gene (TMPRSS2 model shown in Fig. 1 ). In separate reactions, 5' and 3' transcript regions and an endogenous control were amplified by real-time RT-PCR (RNA Ultrasense, Invitrogen; ABI 7900 Sequence Detector, Applied Biosystems).
  • TMPRSS2 IDE In FFPE Tissue: The initial study of 20 FFPE tissue specimens and 42 plasma specimens from patients with prostate cancer or BPH utilized a simple 3':5' ratio cutoff to determined TMPRSS2 Intragenic Differential expression vs. mutual expression of the 2 regions analyzed (Table 1). With a 3':5' ratio cutoff of ⁇ 30, in FFPE tissue, TMPRSS2 IDE was observed in 100% (9/9) prostate cancer specimens and 9% (1/11) BPH specimens. In plasma, early studies yielded 20 samples with RNA passing QC standards.
  • TMPRSS2 IDE was observed in 47% (7/15) PCa, 60% (9/15) PCa samples were positive for 5', 3', or both regions of TMPRSS2, and 20% (1/5) BPH specimens were positive for 1 region of TMPRSS2. Table 1.
  • TMPRSS2 rearrangements by in FFPE tissue Diagnosis TMPRSS2:ERG TMPRSS2 IDE - + ⁇ 0.25 > 0.25 BPH 100% (14/14) 0% (0/14) 71% (10/14) 36% (5/14) Prostate Cancer 44% (14/32) 56% (18/32) 23% (7/31) 84% (26/31) Atypical/PIN 100% (6/6) 0% (0/6) 33% (2/6) 67% (4/6)
  • TMPRSS2 Detection in Plasma Plasma specimens were also assayed for the presence of TMPRSS2:ERG, and 5' UTR and 3' coding regions of TMPRSS2. RNA was extracted from 1 mL (Study 1) or 2 mL (Study 2) plasma from a total of 67 specimens (42 PCa and 17 BPH). Results from samples that sufficiently amplified endogenous control are shown in Table 3. Clearly, Study 2 resulted in a higher rate of detection of 5' UTR or 3' coding region of TMPRSS2 with 78% (7/9) positive PCa and 0% (0/3) BPH as compared to 44% PCa and 17% BPH in Study 1.
  • TMPRSS2 analyzing expression of both 5' UTR and 3' coding regions of TMPRSS2 increases the number of positive specimens by approximately 10-15% compared to detection of 5' UTR or 3' coding region alone and demonstrates significant improvement over detection of TMPRSS2:ERG fusion where only 1 positive PCa specimen was found. Overall, TMPRSS2 was detected in plasma from 44-78% of PCa and 0-17% of BPH specimens. Table 3.
  • ERG and ETV1 IDE in FFPE Tissue The TMPRSS2 IDE strategy was extended to ETS transcription factors.
  • IDE > 0.4 ERG IDE was observed in 97% PCa (29/30), 0% Atypical/PIN (0/5) and 7% BPH (1/14) (Table 4).
  • ETV1 IDE (IDE score > 0.08) was less frequent in PCa, where it was found in 30% (9/30) of specimens, but was also observed 14% BPH (2/14) and 20% Atypical/PIN (1/5).
  • Intragenic differential expression (IDE) of TMPRSS2 as well as ERG are significantly higher in prostate cancer specimens as compared to normal prostate and BPH.
  • IDE Intragenic differential expression
  • the high percentage of PCa specimens with elevated ERG IDE scores may be attributed to translocations with TMPRSS2 as well as other yet to be identified 5' fusion partners such as those recently found to be involved in ETV1 and ETV5 gene fusions, including the 5' UTRs from SLC45A3, HERV-K_22q11.3, C150RF21, and HNRPA2B1 (Helgeson et al. 2008 and Tomlins 2007).
  • ERG IDEs in TMPRSS2 and ETV1 showed unexpected patterns in some samples.
  • ERG IDE was in the orientation of the 3' transcript region being present at higher levels than 5' levels, as would be expected from the understanding that the consequence of TMPRSS2 translocation (and other 5' translocation partners) is an increase in levels of the partnered ETS transcription factor.
  • TMPRSS2 and ETV1 seemed to demonstrate more complexity in that not only were the expected IDE orientations observed, but the reverse orientations were also observed in many samples, meaning that the 5' region of ETV1 and the 3' region of TMPRSS2 were at higher levels than their respective counterpart.
  • TMPRSS2:ETV1 translocations are rarely found when assaying directly for the fusion. Due to these variations, the IDE values are expressed as an absolute value to account for differences in both orientations. Notably, all BPH and Atypical/PIN specimens that were positive for ETV1 IDE demonstrated the reverse orientation while both orientations were observed in prostate cancer samples. Additionally, over one quarter of the prostate cancer samples demonstrated IDE in both ETV1 and ERG. This may be due to the presence of multiple focal points or multiple clonalities in a single specimen. It is apparent however, that ERG IDE is observed primarily in confirmed prostate cancer and was only observed in one BPH specimen.
  • a fusion gene with transforming activity, echinoderm microtubule associated protein like 4 - anaplastic lymphoma kinase is found in approximately 5% of NSCLCs of lung cancer patients.
  • the presence of the EML4-ALK fusion can be predictive of the response of these patients to certain therapies.
  • We applied the IDE methodology to test the ability of using IDE to identify patients with potential ALK translocation, not limited to know variants or fusion partners.
  • Patient lung cancer tissue samples were analyzed using the IDE methodology by determining the ALK IDE cutoff value and then comparing the calculated cutoff to the ALK IDE values from lung cancer tissue samples.
  • the positive results were further analyzed by direct detection of EML4-ALK fusions using RT-PCR.
  • a subset of the NSCLC positive samples were screened by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH).
  • This ALK IDE cutoff value of .7 was subsequently used to identify abnormal ratios in tissue specimens, indicating the presence of ALK rearrangement. Further verification of the methodology confirmed that the ALK IDE value was 0.0 in two EML4-ALK negative NSCLC cell lines (NCI-H838 and NCI-H1299).
  • ALK Amplification Primers and Probes for IDE Analysis 3' ALK Primers 3' ALK Forward CCCAACTTTGCCATCATTTT (SEQ ID NO: 13) 3' ALK Reverse GCAAAGCGGTGTTGATTACA (SEQ ID NO: 14) 3' ALK Probe FAM-TGAATACTGCACCCAGGACC-BHQ (SEQ ID NO: 15) 5' ALK Primers 5' ALK Forward TGGCTTTTGACAATATCTCCA (SEQ ID NO: 16) 5' ALK Reverse TGCAGGATCTTGTCCTCTCC (SEQ ID NO: 17) 5' ALK Probe AGCCTGGACTGCTACCTCAC (SEQ ID NO: 18)
  • ALK IDE in Lung Cancer Tissue Using the ALK IDE 3':5' ratio cutoff of >0.7, in EML4-ALK fusion-positive cell lines, a diagnostically positive ALK IDE was observed in 11% (6/56) of lung cancer tissue specimens.
  • Immunohistochemistry (IHC) and / or fluorescence in situ hybridization (FISH) A subset of the NSCLC positive samples were screened by IHC and/or FISH (subset results are shown in Fig. 7 ). The five confirmed ALK IDE positive samples and an additional five samples with slightly to moderate elevated levels of ALK transcript (i.e., ALK IDE > 0.1) were further analyzed by FISH. Eight of the ten samples showed ALK rearrangement and/or ALK gene amplification. Three samples interpreted as having ALK rearrangements by FISH were also positive using ALK IDE. Two other ALK IDE positive samples (one confirmed and one unconfirmed by direct RT-PCR) were interpreted as rearrangement negative by FISH.
  • IHC immunohistochemistry
  • FISH fluorescence in situ hybridization

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EP4119675A1 (fr) 2023-01-18
US20190093176A1 (en) 2019-03-28
US8426133B2 (en) 2013-04-23
EP3133168B1 (fr) 2019-01-23
US20130316341A1 (en) 2013-11-28
US20150315658A1 (en) 2015-11-05
US20170152572A1 (en) 2017-06-01
EP2435584A1 (fr) 2012-04-04
EP3546596B1 (fr) 2022-05-11
US9546404B2 (en) 2017-01-17
US9187788B2 (en) 2015-11-17
CA2763608A1 (fr) 2010-12-02
US10501809B2 (en) 2019-12-10
US11021758B2 (en) 2021-06-01
CA2763608C (fr) 2020-12-29
US8815516B2 (en) 2014-08-26
EP2435584B1 (fr) 2016-07-13
US20150038367A1 (en) 2015-02-05
US20100304390A1 (en) 2010-12-02
CN108359719A (zh) 2018-08-03
EP2435584A4 (fr) 2013-04-03
WO2010138460A1 (fr) 2010-12-02
US20200181718A1 (en) 2020-06-11

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